Brain ischemia caused by cardiac arrest and stroke is a significant source of human morbidity and mortality. This proposal focuses on the role of calpains, a family of Ca2+-dependent cytosolic proteases, in delayed necrosis of post-ischemic neurons. Brain calpain activity is pathologically increased after brain ischemia, and calpain inhibitors are neuroprotective in preclinical models. However, the mechanism of calpain-mediated injury is unknown, and the relative roles of the two ubiquitous isoforms, u-calpain and m-calpain, have not been elucidated. Pathologic calpain activity requires sustained cytosolic Ca2+ elevation. Calpain-mediated cleavage of IP3 receptors (IP3R) and ryanodine receptors (RYR) generates stable dysregulated channels that have increased Ca2+ conductance. These observations support the hypothesis that calpains are not only activated by elevated cytosolic Ca2+, but under pathologic conditions contribute to sustained cytosolic Ca2+ overload in a potentially irreversible feed-forward pathway that ultimately causes neuronal necrosis. Specific Aim 1 will use in vivo adeno-associated viral (AAV) vector-mediated overexpression of the specific endogenous calpain inhibitor, calpastatin, to elucidate the causal relationship between post-ischemic calpain activity, cytosolic Ca2+ overload, electrophysiological dysfunction, and delayed necrosis of hippocampal CA1 pyramidal neurons in a rat model of transient forebrain ischemia. In Specific Aim 2, the relative role of u-calpain and m-calpain will be examined using AAV vector-mediated RNA interference in the same model. Specific Aim 3 will examine the role of calpain-cleaved IP3R1. Generation of calpain-cleaved IP3R1 in post ischemic neurons will be immunohistochemically characterized. A truncated IP3R1 mutant corresponding to the stable calpain-derived fragment will be expressed in Xenopus oocytes for nuclear patch clamp analysis of channel function and expressed in CA1 pyramidal neurons in vivo to determine if it causes calpain activity and delayed necrosis. Specific Aim 4 will utilize a similar approach to evaluate the role of calpain-cleaved RYR2. The proposed studies overcome significant obstacles limiting the mechanistic evaluation of calpain's role in in vivo ischemic brain injury. The results will provide fundamental insights into the mechanism of delayed post-ischemic neuronal necrosis, and facilitate the development of effective therapies for patients suffering from cardiac arrest and stroke.